Control of membrane sealing in injured mammalian spinal cord axons

The process of sealing of damaged axons was examined in isolated strips of white matter from guinea pig spinal cord by recording the "compound membran e potential," using a sucrose-gap technique, and by examining uptake of hor seradish peroxidase (HRP). Following axonal transection, exponential recove ry of membrane potential occurred with a time constant of 20 +/- 5 min, at 37 degrees C, and extracellular calcium activity ([Ca2+](o)) of 2 mM. Most axons excluded HRP by 30 min following transection. The rate of sealing was reduced by lowering calcium and was effectively blocked at [Ca2+](o) less than or equal to 0.5 mM, under which condition most axons continued to take up HRP for more than 1 h. Sealing at higher [Ca2+](o) was blocked by calpa in inhibitors (calpeptin and calpain inhibitor-1) indicating a requirement for type II (mM) calpain in the sealing process. Following compression inju ry, the amplitude of the maximal compound action potential conducted throug h the injury site was reduced. The extent of amplitude reduction was increa sed when the tract was superfused with calcium-free Krebs' solution (Ca2+ r eplaced by Mg2+). These results suggest that the fall in [Ca2+](o) seen fol lowing injury in vivo is sufficient to prevent membrane sealing and may par adoxically contribute to axonal dieback, retrograde cell death, and "second ary" axonal disruption.